HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/11/1264 03-1232fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by CHAI, J. Articles by TARNAWSKI, A. S. Search for Related Content PubMed PubMed Citation Articles by CHAI, J. Articles by TARNAWSKI, A. S.

Serum response factor is a critical requirement for VEGF signaling in endothelial cells and VEGF-induced angiogenesis

Department of Medicine/Gastroenterology, VA Medical Center, Long Beach, California, USA; and the University of California, Irvine, California, USA

¹Correspondence: Gastroenterology Section (111G), VA Medical Center, 5901 E. Seventh St., Long Beach, CA 90822, USA. E-mail: atarnawski{at}yahoo.com

SPECIFIC AIMS

Angiogenesis is a process of new blood microvessel formation and is regulated by angiogenic growth factors such as vascular endothelial growth factor (VEGF). Angiogenesis plays essential roles in embryonic development, wound healing, and tumor growth. Serum response factor (SRF) is a transcription factor required for embryogenesis and muscle development and function. Its role in endothelial cell biology and angiogenesis has not been explored, nor has the effect of VEGF on SRF activation. This study was aimed to determine the role of SRF in VEGF-activated endothelial cell migration and proliferation and in vitro and in vivo angiogenesis; and to explore the effect of VEGF on SRF expression and activation.

PRINCIPAL FINDINGS

1. SRF is required for VEGF-induced in vitro angiogenesis
To determine whether SRF is required for VEGF-induced angiogenesis, we introduced SRF antisense oligonucleotides into human umbilical vein endothelial cells (HUVEC) and rat gastric microvascular endothelial cells (RGMEC) to knock down SRF protein expression. In the SRF antisense oligo-treated HUVEC cells (aSRF), SRF protein levels were reduced by 92 ± 6% (P<0.001) at 48 h after treatment (Fig. 1 A) compared with vehicle-treated (control) or SRF sense oligo-treated cells (sSRF), indicating aSRF-induced SRF deficiency. A significant reduction (68±5%; P<0.001) in SRF protein expression was also obtained in aSRF RGMEC cells. Treatment with SRF antisense oligonucleotide dramatically inhibited VEGF-induced capillary-like structure formation by 88 ± 3% and 84 ± 5% in HUVEC and RGMEC cells (both P<0.001), respectively, as reflected by 2-D Matrigel assays (Fig. 1B, D ). This treatment inhibited HUVEC cell sprouting by 95 ± 5% (P<0.001) in 3-D collagen gels (Fig. 1C, D ).

View larger version (55K): [in this window] [in a new window]   Figure 1. SRF is a critical requirement for VEGF-induced in vitro angiogenesis in HUVEC cells. A) Western blot analyses showing that treatment with SRF antisense oligonucleotide (aSRF) suppressed SRF protein expression in HUVEC cells whereas treatment with the complementary sequence (sSRF), as a control, did not significantly alter SRF protein expression levels. B) SRF-deficient HUVEC cells (aSRF) failed to form capillary-like structures on 2-D Matrigels in response to VEGF treatment. C) VEGF treatment also failed to stimulate sprouting in SRF-deficient HUVEC cells (aSRF) within 3-D collagen gel matrices. D) Quantitative analyses showing that treatment with SRF antisense oligonucleotide in HUVEC cells (aSRF) caused an 88 ± 3% reduction in VEGF-induced capillary-like tube formation on 2-D Matrigels and a 95 ± 5% reduction in the number of sprouting cells within 3-D collagen gel matrices.

Taken together, these data indicate that SRF is a critical requirement for VEGF-induced in vitro angiogenesis.

2. SRF deficiency inhibits VEGF-stimulated endothelial cell migration
Since cell migration is essential for angiogenesis, SRF deficiency might have impaired VEGF-induced in vitro angiogenesis by inhibiting endothelial cell migration. To test this, we wounded endothelial cell monolayers by making standardized excisions with a razor blade and counted the number of cells that migrated into the denuded area. Forty-eight hours after wounding, the aSRF RGMEC cells had a >4-fold reduction (P<0.001) in migration compared with sSRF or control cells. Cell staining revealed an inhibition of VEGF-induced actin polymerization in the aSRF cells.

3. SRF deficiency impairs VEGF-stimulated endothelial cell proliferation
Endothelial cell proliferation is another important component of angiogenesis. ³H-Thymidine incorporation assays demonstrated that after 24 h incubation with VEGF, SRF-deficient RGMEC cells had a 42 ± 5% reduction (P<0.001) in the amount of thymidine incorporated compared with sSRF or control cells, indicating that SRF is important for VEGF-induced endothelial cell proliferation. VEGF failed to activate c-Fos or Egr-1 expression (both are important for cell proliferation) in the SRF-deficient endothelial cells.

4. VEGF activates SRF expression in endothelial cells
Inhibition of VEGF-induced in vitro angiogenesis as a result of SRF deficiency suggests that SRF is a critical effector of VEGF-induced angiogenic signaling in endothelial cells. To define the effect of VEGF on SRF activation, we sequentially examined expression of SRF mRNA and protein in HUVEC and RGMEC cells after VEGF treatment. In both types of cells, VEGF treatment significantly increased SRF mRNA by 3-fold at 1 h and increased protein levels by 7-fold between 2 and 6 h (both P<0.01). This result clearly demonstrates that VEGF is a potent inducer of SRF mRNA and protein expression in endothelial cells. VEGF stimulated SRF nuclear translocation and DNA binding activity, as demonstrated by SRF immunocytochemical staining and EMSA, respectively, indicating that VEGF induces both the expression and activation of SRF.

5. Activation of SRF in endothelial cells by VEGF requires both MEK-ERK and Rho signaling
To gain a mechanistic understanding of how VEGF activates SRF, we pretreated HUVEC and RGMEC cells for 30 min with the MEK-specific inhibitors U0126 or PD98059, then treated the cells with VEGF for 30, 60, and 120 min. Blocking ERK activation by these pretreatments abolished the VEGF-induced increase in SRF protein expression in both types of cells (Fig. 2 A).

View larger version (57K): [in this window] [in a new window]   Figure 2. SRF activation by VEGF in RGMEC cells requires both MEK-ERK and Rho-actin signaling. A) Western blot analyses showing that VEGF failed to activate SRF expression in RGMEC cells when MEK-ERK signaling was inhibited by U0126. B) Western blot analyses showing that VEGF failed to activate SRF expression in RGMEC cells when Rho signaling was inhibited by Clostridium difficile toxin B. C) Western blot analyses showing that VEGF failed to activate SRF expression in RGMEC cells when actin polymerization was inhibited by latrunculin B. D) Fluorescence double staining of RGMEC cells confirming that F-actin polymerization and stress fiber formation are inhibited by latrunculin B. E) Quantitative analyses showing a strong correlation between F-actin/G-actin ratio and SRF expression. F) Inhibition of actin polymerization by latrunculin B in RGMEC cells prevented VEGF-induced in vitro angiogenesis as reflected by 2-D Matrigel and 3-D collagen gel assays.

To determine whether Rho GTPases were required for VEGF-induced SRF expression, we pretreated HUVEC and RGMEC cells for 60 min with Clostridium difficile toxin B, a specific inhibitor of Rho GTPases, then incubated the cells with VEGF for 30, 60, and 120 min. Pretreatment with toxin B abolished VEGF-induced SRF protein expression in endothelial cells (Fig. 2B ).

6. Inhibition of actin polymerization blocks VEGF-induced SRF activation in endothelial cells and VEGF-induced in vitro angiogenesis
Recent studies indicate that SRF activation in NIH 3T3 cells is induced through depletion of cellular G-actin pool. Since VEGF induces formation of actin stress fibers in endothelial cells, which in turn leads to depletion of the G-actin pool, we tested whether VEGF activates SRF through a mechanism involving actin polymerization. HUVEC and RGMEC cells were pretreated for 60 min with an actin polymerization inhibitor, latrunculin B, then incubated with VEGF. Latrunculin B pretreatment significantly inhibited VEGF-induced SRF expression in both types of endothelial cells (Fig. 2C ). Latrunculin B-inhibited actin polymerization was confirmed by G- and F-actin double staining (Fig. 2D ). There was a strong correlation (r=0.87; P<0.01) between the ratio of cellular F-actin to G-actin and SRF protein levels (Fig. 2E ), indicating that actin polymerization promotes SRF expression in the endothelial cells. Latrunculin B pretreatment inhibited VEGF-induced SRF nuclear translocation and reduced VEGF-induced SRF DNA binding activity in endothelial cells. Latrunculin B-pretreated HUVEC and RGMEC cells failed to form either tubular structure on 2-D Matrigel or sprouts in 3-D collagen gel matrix in response to VEGF, reflecting inhibition of angiogenesis in vitro (Fig. 2F ).

7. SRF deficiency inhibits angiogenesis in vivo
Angiogenesis is essential for wound healing, as tissue regeneration requires restoration of the microvascular network to provide oxygen and nutrient delivery to the healing site. To determine whether SRF is also important for angiogenesis in vivo in a physiologically/pathologically relevant condition, we used an experimental gastric ulcer model. Rats with experimentally induced gastric ulcers were injected with a plasmid that expresses SRF antisense RNA to knock down SRF protein levels in the ulcer area. SRF antisense RNA expression was detectable in the ulcer granulation tissue from day 3 to 7 postinjection, and this corresponded to significantly suppressed SRF protein levels. Local SRF deficiency significantly reduced the number of regenerating microvessels in granulation tissue at the ulcer base (7±2/ mm² in the aSRF plasmid-injected rats compared with 32±5/mm² in controls; P<0.01), reflecting inhibited angiogenesis.

CONCLUSIONS AND SIGNIFICANCE

This study demonstrates that SRF is a critical requirement for VEGF-induced in vitro angiogenesis and endothelial cell migration, proliferation, and actin cytoskeleton rearrangements. VEGF induces SRF expression, nuclear translocation, and DNA binding activity in endothelial cells. This activation requires actin polymerization, MEK-ERK, and Rho GTPase signaling, since blocking these pathways with specific inhibitors resulted in abolishment of VEGF-induced SRF activation.

A new transcription factor, MAL, was recently discovered in NIH 3T3 cells to be the critical sensor of cellular G-actin levels. In quiescent NIH3T3 cells, MAL is predominantly localized to the cytoplasm, where it is sequestered by G-actin. Upon cell stimulation by mitogens, G-actin dissociates from MAL and polymerizes into F-actin. MAL is liberated from G-actin and translocates into nucleus, where it forms an association with SRF to activate SRE-dependent genes. Here we demonstrated that VEGF stimulates actin stress fiber formation in endothelial cells. When this process is blocked by specific inhibitors such as latrunculin B, VEGF loses its ability to induce SRF activation and angiogenesis. SRF deficiency in endothelial cells results in severe impairment of VEGF-induced cell migration and proliferation, suggesting that SRF is critical to most, if not all, processes regulated by VEGF.

View larger version (25K): [in this window] [in a new window]   Figure 3. Diagram depicting a central role of SRF in VEGF-induced angiogenesis. VEGF stimulates actin polymerization through Rho signaling, which is prevented by the Rho inhibitor Clostridium difficile toxin B and the Rho kinase inhibitor Y27632. Actin polymerization leads to G-actin depletion resulting in SRF activation, which is prevented by the actin polymerization inhibitor, latrunculin B. VEGF stimulates the Ras-MEK-ERK pathway that leads to Sp1 activation, critical for SRF activation. This pathway is blocked by MEK inhibitors U0126 or PD98059. SRF activation promotes cell migration through the regulation of actin dynamics and other cytoskeletal genes and promotes cell proliferation through activation of immediate-early genes. Both endothelial cell migration and proliferation are important for angiogenesis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1232fje; doi: 10.1096/fj.03-1232fje

This article has been cited by other articles:

				A. M. Verdoni, N. Aoyama, A. Ikeda, and S. Ikeda Effect of destrin mutations on the gene expression profile in vivo Physiol Genomics, June 10, 2008; 34(1): 9 - 21. [Abstract] [Full Text] [PDF]

				M.-R. Du, W.-H. Zhou, L. Dong, X.-Y. Zhu, Y.-Y. He, J.-Y. Yang, and D.-J. Li Cyclosporin A Promotes Growth and Invasiveness In Vitro of Human First-Trimester Trophoblast Cells Via MAPK3/MAPK1-Mediated AP1 and Ca2+/Calcineurin/NFAT Signaling Pathways Biol Reprod, June 1, 2008; 78(6): 1102 - 1110. [Abstract] [Full Text] [PDF]

				J. Chai, M. Norng, A. S Tarnawski, and J. Chow A critical role of serum response factor in myofibroblast differentiation during experimental oesophageal ulcer healing in rats Gut, May 1, 2007; 56(5): 621 - 630. [Abstract] [Full Text] [PDF]

				J. M. Miano, X. Long, and K. Fujiwara Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus Am J Physiol Cell Physiol, January 1, 2007; 292(1): C70 - C81. [Abstract] [Full Text] [PDF]

				N. F. Voelkel, R. W. Vandivier, and R. M. Tuder Vascular endothelial growth factor in the lung Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L209 - L221. [Abstract] [Full Text] [PDF]

				B. Camoretti-Mercado, N. O. Dulin, and J. Solway SRF Function in Vascular Smooth Muscle: When Less is More? Circ. Res., September 2, 2005; 97(5): 409 - 410. [Full Text] [PDF]

				A. V. Ljubimov and M. B. Grant P450 in the Angiogenesis Affair: The Unusual Suspect Am. J. Pathol., February 1, 2005; 166(2): 341 - 344. [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/11/1264 03-1232fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by CHAI, J. Articles by TARNAWSKI, A. S. Search for Related Content PubMed PubMed Citation Articles by CHAI, J. Articles by TARNAWSKI, A. S.